This project investigates signal transduction pathways that regulate proliferation, migration, and differentiation of lens and corneal epithelial cells with the goal of expanding the basic understanding of these pathways and identifying enzymes that may be therapeutically targeted in pathological conditions. The primary concentration in this fiscal year has been to investigate the role of the proline-directed kinase, Cdk5, in regulating cell-cell and cell-matrix adhesion. One avenue of investigation has been to identify lens proteins that interact with Cdk5 or its activating proteins, p35 and p39, using the yeast two-hybrid technique. These studies have identified two proteins, muskelin and myosin essential light chain, as specific interactors of p39. Both of these proteins are involved in cytoskeletal regulation, and may participate in Cdk5-dependent regulation of cell-matrix or cell-cell adhesion. Studies that were in progress last fiscal year to determine the relationship between the Cdk5 activating protein, p39, and the beta-propeller protein, muskelin, were completed and published in this year. The results demonstrated that a unique insert in the C-terminus of p39 (which is not present in the closely related Cdk5 activating protein, p39) contains a strong binding site for muskelin. Moreover, co-expression of muskelin and p39 was shown to shift the intracellular localization of muskelin to the peripheral actin cytoskeleton. This finding suggests that p39 may be responsible for linking muskelin to the cytoskeleton and may thus, play an integral role in muskelin-dependent effects on cell adhesion. Studies of the p39-myosin essential light chain (MLC) interaction also made considerable progress this fiscal year. We have shown that MLC binds the N-terminus of p39, raising the possibility that p39 may link muskelin to the cytoskeleton via attachment to MLC. Co-immunoprecipitation results are consistent with this possibility and additional, more stringent, tests are in progress. Previous studies of corneal epithelial wound closure indicated that inhibitors of Cdk5 enhance the rate of epithelial cell migration, suggesting that such inhibitors might be therapeutically useful in treating certain corneal epithelial defects. This possibility is being pursued in an ongoing collaboration with Dr. Mary Ann Stepp (GWU). Preliminary tests to determine the best means of delivery and to assess the safety and efficacy of Cdk5 inhibitors in promoting corneal wound closure in mice are in progress. Analysis has proceeded on transgenic mice generated in the previous fiscal year that overexpress Cdk5 or a kinase inactive form of Cdk5 only in lens fibers under the direction of the betaB1 crystallin promoter. Preliminary analysis of these animals had suggested that Cdk5 promotes cell-cell adhesion of lens fibers, and this observation has been confirmed by additional studies. We have shown that the fraction of total N-cadherin that is associated with the actin cytoskeleton (and therefore, engaged in productive cell-cell adhesions) is significantly lower in lens fiber cells expressing kinase inactive Cdk5. Studies of the role of Cdk5 in regulating E-cadherin junction formation in cornea have led to a similar conclusion. We have found that suppressing Cdk5 expression in cultured corneal epithelial cells with siRNA leads to a reduction in the fraction of E-cadherin associated with cell-cell boundaries in an intact epithelial sheet. Moreover, pharmacological inhibition of Cdk5 activity reduces the ability of dissociated corneal epithelial cells to attach to a surface coated with E-cadherin. Thus, Cdk5 seems to inhibit formation of both E- cadherin- and N-cadherin-dependent junctions. To examine the mechanism of this effect, experiments are in progress to analyze the role of Cdk5 in endocytotic recycling of cadherin, using surface biotinylation to distinguish surface and internalized cadherin. Since our previous work had shown that Cdk5 may exert some of its effects by modulating Src activation, we have undertaken studies to examine the relationship between Src and Cdk5 in cornea and lens. Results obtained this fiscal year indicate that these two kinases directly interact and have mapped the binding site to the N-terminal 100 amino acids of Cdk5. Experiments are in progress to determine the consequences of this interaction for cell-matrix and cell-cell adhesion. In addition, we are continuing our collaboration with Dr. A.S. Menko, (Thomas Jefferson University) to examine whether Src family kinases play a role in the Cdk5-dependent modulation of N-cadherin junctions in the lens.